最近，一項稱為階層寡核甘酸引子延伸(Hierarchical Oligonucleotide Primer Extension, HOPE)新型定性定量分子生物技術的成熟發展，使我們可以針對環境樣本中複雜的微生物社群結構進行高通量的分析。本研究為了建立甲烷菌群結構的HOPE高通量分析平台，根據16S rRNA序列的親緣分析，將已知31屬之甲烷菌在不同的分類階層進行專一性的核甘酸引子(oligoprimer)設計，經測試與最佳化實驗條件共成功獲得28個引子，配裝成7組的多通量分析引子套組，每套分析的通量數可達4-6個目標甲烷菌群。以建立的HOPE平台三重覆分析30個反應槽的厭氧污泥樣本，經22小時的HOPE實驗後，可快速獲知這些樣本中偵測的5個目與23個屬甲烷菌群的相對豐富度數據，而且引子偵測專一性高，分析標準差小於12.2 %，靈敏度低達0.165 %。結果顯示這30種處的厭氧污泥中嗜醋酸及胺類化合物上甲基團的Methanosarcinales目喜好棲息於厭氧污泥環境， 其豐富度在大部份的反應槽污泥中均佔79.3 %以上，其中嗜醋酸的Methanosaeta屬及Methanosarcina屬個別優勢於不同的反應槽污泥。此外，偵測到Methanomethylovorans屬甲烷菌的普遍存在，其相對豐富度達3.85~71.5 %，尤其優勢於處理TFT-LCD廢水反應槽的污泥，顯示是廢水厭氧處理重要的甲烷菌群。另一方面，偵測到的嗜氫甲烷菌有Methanomicrobiales目(6.1~52.5 %)、Methanobacteriales目(3.3~88.7 %)等。由上述結果可知，本研究發展出的HOPE平台可有效地進行反應槽污泥中甲烷菌群結構的定性定量分析，如與其他分生技術如cloning&sequencing、定量PCR搭配運用可快速提供改進反應槽操控所需甲烷菌群多樣性的資訊。本研究建立的HOPE技術與分析策略可推廣應用於研究不同厭氧生物環境中甲烷菌的生態。

In this study, a newly developed molecular method called hierarchical oligonucleotide primer extension, HOPE was optimized to qualitatively and qualitatively analyze the methanogenic Archaea in the sludge from anaerobic reactors treating industrial wastewaters. In total, 28 oligonucleotide primers with specificity at the various hierarchical levels were designed and successfully applied in 7 multiplexing analyses. The HOPE multiple analysis achieved a simultinuous detection of 4-6 targeting methanogenic archaea. After optimization, the assay was used to detect the targeting methanogens in 30 different sludge samples, which triplicate analysis could be completed within xx mins, coming up with the abundance values of 5 orders and 23 genera methanogens with excellent detection specificity, reproducibility (std < 12.2 %) and sensitivity (~0.165 %). The results showed that the detected methanogens by the developed HOPE assay could cover a total abundance of at least 79.3 % in the domain Archaea. It was further observed that the methanogenic populations in the order Methanosarcinales were highly abundant, in which members in the genera of Methanosaeta (<101%)、Methanosarcina (0.8~22.4 %) or Methanomethylovorans (3.85~71.5 %) were the predominant methanogenic populations in the respective anaerobic sludge from bioreactors, showing their important roles in the anaerobic treatment systems. Furthermore, the detected hydrogenotrophic methanogens were Methanomicrobiales(6.1~52.5 %)、Methanobacteriales(3.3~88.7 %).Overall, the established HOPE assay can effectively quantify the methanogens in engineered ecosystems, hence providing valuable information to better improve the performance of anaerobic bioreactors. The encourageing conclusion further suggests that this platform developed in this study may be applicable for samples from other environments.

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